HiPerFect HTS Reagent

Please note: The HiPerFect HTS Reagent range has been discontinued (cat. nos. 301802, 301806, 301807). As an alternative, we recommend using the HiPerfect Transfection Reagent portfolio, which can be used to transfect the same cell types.

The HiPerFect HTS Reagent is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

HiPerFect HTS Reagent outperforms alternative reagent.

HCT116 cells were transfected with CDC2 siRNA or AllStars Negative Control siRNA at the concentrations indicated in 96-well plates. Transfections were performed using HiPerFect HTS Reagent or Reagent R from an alternative supplier. Gene silencing was analyzed by real-time, quantitative RT-PCR. Knockdown level is shown on the left y-axis. Samples transfected using HiPerFect HTS Reagent demonstrated consistently high CDC2 knockdown for all siRNA concentrations tested. In contrast, samples transfected using Reagent R provided less effective knockdown at lower siRNA concentrations. Analysis of cytotoxicity was also performed using the CellTiter-Glo Assay (Promega) after siRNA transfection of AllStars Negative Control siRNA at the concentrations indicated. Percentage survival relative to untransfected cells is shown as red dots and on the right y-axis. Hi PerFect HTS Reagent showed a high rate of cell survival. Lower cell survival levels were observed after transfection using Reagent R.

Efficient transfection in human and mouse cells.

Various cell types were transfected with Lamin A/C siRNA or AllStars Negative Control siRNA at the concentrations indicated using HiPerFect HTS reagent in 384-well plates. Gene silencing was analyzed by real-time, quantitative RT-PCR. HiPerFect HTS Reagent provided high Lamin A/C knockdown for all concentrations tested in all cell types.

High lot-to-lot consistency.

HeLa S3 cells were transfected with CDC2 siRNA or AllStars Negative Control siRNA at the concentrations indicated using HiPerFect HTS Reagent from 5 different lots in 96-well plates. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with CDC2 siRNA demonstrated consistently high CDC2 knockdown for all reagent lots.

High stability in dilution.

HeLa S3 cells were transfected with Lamin A/C siRNA (5 nM) or AllStars Negative Control siRNA (5 nM) using HiPerFect HTS Reagent in 96-well plates. After dilution in culture media, HiPerFect HTS Reagent was either used immediately for transfection, or stored at room temperature for 6 hours and then used for transfection. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with Lamin A/C siRNA demonstrated consistently high Lamin A/C knockdown for both freshly diluted HiPerFect HTS Reagent and HiPerFect HTS Reagent stored for 6 hours at room temperature.

Effective transfection at a range of cell densities and siRNA concentrations.

HepG2 cells were transfected with Lamin A/C siRNA or AllStars Negative Control siRNA at the concentrations indicated using 0.1 µl HiPerFect HTS Reagent per sample in 96-well plates. Cell density at the time of transfection is indicated. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with Lamin A/C siRNA demonstrated high Lamin A/C knockdown.

Reproducibly high transfection and silencing efficiency.

MCF-7 cells were transfected with CDC2 siRNA (10 nM, 20 nM, and 30 nM) or AllStars Negative Control siRNA (30 nM) using HiPerFect HTS reagent in 96-well plates. Each transfection was performed in triplicate. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with CDC2 siRNA demonstrated consistently high CDC2 knockdown in all replicates.

High-throughput transfection using HiPerFect HTS Reagent.

Performance

In high-throughput transfection, high numbers of samples are processed simultaneously. The use of a robust, high-performing reagent is essential for a standardized, smooth-running workflow. HiPerFect HTS Reagent offers many benefits, including high lot-to-lot consistency and high stability in dilution (see figure "High lot to lot consistency"). These qualities minimize experimental artifacts and make experimental setup easier. For example, HiPerFect HTS Reagent can be diluted in the morning and used for complex formation throughput the day without any decrease in performance (see figure "High stability in dilution"). HiPerFect HTS Reagent delivers high transfection and silencing efficiency across a range of siRNA concentrations (see figure "Reproducibly high transfection and silencing efficiency"). In addition, HiPerFect HTS Reagent consistently provides high transfection efficiency if variations in cell density occur (see figure "Effective transfection at a range of cell densities and siRNA concentrations"). HiPerFect HTS Reagent can be kept at room temperature during use and is stable for 2 years after receipt.

HiPerFect HTS Reagent is specially optimized for use in a high-throughput experimental setup. Reliable results are assured in a wide range of cell lines, including primary cells (see table; and figure "Efficient transfection in human and mouse cell lines"). Protocols for transfection of cells in 96-well and 384-well plates are provided.

Cell lines successfully transfected using HiPerFect HTS Reagent

Human adherent cell lines

Human primary cells

Mouse adherent cell lines

293, HCT116, LNCaP, A549, HeLa, HeLa S3

HUVEC

NIH/3T3

AGS, HepG2, MCF7, Caco2, Huh7, MDA MB231

NHDF

B16F1

Principle

Efficient transfection of siRNA is critical for effective gene silencing. High-throughput transfection conditions must be highly optimized and standardized to minimize artifacts and maximize reproducibility. Transfections should be carried out using low siRNA concentrations and a reagent that ensures low cytotoxicity. HiPerFect HTS Reagent has been developed for efficient transfection of cells even when low siRNA concentrations are used. In addition, cells transfected using HiPerFect HTS Reagent show high cell viability and low cytotoxicity provoding reliable and accurate results.

miRNA research

microRNAs (miRNAs) are a class of endogenous small RNA molecules with similar characteristics to siRNAs. miRNAs play a role in many diverse biological processes such as development, differentiation, and apoptosis. Transfection of synthetic miRNA mimics or inhibitors is a technique used to elucidate the targets and roles of particular miRNAs. miRNA mimics are chemically synthesized miRNAs which mimic naturally ocurring miRNAs upon transfection into the cell. miRNA inhibitors are single-stranded modified RNAs which specifically inhibit miRNA function. Reduced gene expression after transfection of an miRNA mimic, or increased expression after transfection of an miRNA inhibitor, provide evidence that the targeted miRNA is involved in regulation of that gene. Alternatively, the role of miRNAs in various pathways can be studied by examination of a specific phenotype after transfection of an miRNA mimic or inhibitor. Screening experiments can be performed to examine a phenotype after transfection of large numbers of miRNA mimics or inhibitors. HiPerFect HTS Reagent was developed for highly efficient transfection of eukaryotic cells with siRNA, and is ideally suited to transfection of miRNA mimics or inhibitors.

Procedure

In high-throughput experiments, reductions in time-to-result, labor, and consumable use significantly improve the workflow. Using HiPerFect HTS Reagent, cells are seeded and transfected on the same day, saving time and effort (see figure "High-throughput transfection using HiPerFect HTS Reagent"). Just one plate is used for both complex formation and transfection, minimizing consumable use and pipetting steps. In addition to saving on plastic consumables, low volumes of HiPerFect HTS Reagent are used for each transfection, ensuring maximum cost-efficiency without compromising on reproducibility and reliability of results (see figure "HiPerFect HTS Reagent outperforms alternative reagent").